Soluble melanin

ABSTRACT

A melanin that is soluble in an aqueous solution at a pH of at least 5 to 9 at a temperature of 0° to 100° C. The melanin is further characterized by being capable of being filtered through at least a 0.45 micron size filter. Still further, the melanin is characterized by having a molecular weight of greater than 10,000 kilodaltons. The melanin is useful for providing a naturally-appearing tan to mammalian skin and hair. Such melanin can be produced by combining dopachrome and 5,6-dihydroxyindole (or allowing dopachrome to spontaneously form 5,6-dihydroxyindole) and an appropriate enzyme or by combining 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid or by incubating 5,6-dihydroxyindole-2-carboxylic acid alone. The melanin is also useful for providing a sun-screen to mammalian skin and hair.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 603,111, filed Oct. 25, 1990now U.S. Pat. No. 5,218,079 which is a CIP of Ser. No. 525,944, filedMay 18, 1990, now U.S. Pat. No. 5,216,116.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns the synthesis of soluble forms of melaninand their composition, and methods of using such compositions to providea naturally-appearing tan to mammalian skin and hair and to provide asun-screen.

2. Background Information

In biology, melanins are heteropolymers consisting of L-dopa and itsenzymatic derivatives. They are ubiquitous in living organisms and areproduced throughout the zoological and botanical phyla. In mammalianskin, melanins are produced through enzymatic processes in specializedcells known as "melanocytes". Melanins are the pigments of mammalianskin and hair.

Mammalian melanins are highly insoluble and can be dissolved(solubilized) only through non-physiological treatments such as boilingin strong alkali, or through the use of strong oxidants such as hydrogenperoxide. Tyrosinase, a key enzyme in the melanin biosynthetic pathway,can catalyze the formation of melanin in a test tube using L-tyrosineL-dopa or 5',6'-dihydroxyindole as substrates, however, the product isan insoluble precipitate as described above.

Ito, "Reexamination of the Structure of Eumelanin", Biochimica etBiophysica Acta, 883, 155-161, 1986, mentions natural melanin may be apolymer of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylicacid. Ito, however, does not teach or suggest combining these chemicalsto form melanin.

Ito and Nicol, "Isolation of Oligimers of5,6-Dihydroxyindole-2-carboxylic Acid from the Eye of the Catfish",Biochemical Journal, 143, 207-217, 1974, mention that oligimers of5,6-dihydroxyindole-2-carboxylic acid exist in nature, for example inthe tapetum lucidum of the sea catfish (Arius felis). Ito and Nicol,however do not teach or suggest that these structures could be used as aform of soluble melanin.

Palumbo, d'Ischia, Misuraca, and Prota, "Effect of metal ions on therearrangement of dopachrome", Biochimica et Biophysica Acta, 925,203-209, 1987, mention that the metal ions CU²⁺, Ni²⁺, and CO²⁺ areeffective in inducing the non-decarboxylative rearrangement ofdopachrome at physiological pH values, leading mainly to the formationof 5,6-dihydroxyindole-2-carboxylic acid. They suggest that whenconsidered in the light of the known metal accumulation in pigmentedtissues, their results provide a new entry into the regulatorymechanisms involved in the biosynthesis of melanins. Palumbo et al,however, do not teach or suggest that such metal ions could be used toaffect the color or formation of soluble melanin. Likewise, Leonard,Townsend, and King, "Function of Dopachrome Oxidoreductase and MetalIons in Dopachrome Conversion in the Eumelanin Pathway", Biochemistry,27, 6156-6159, 1988, present similar results to those of Palumbo et alregarding metal ions and the formation of5,6-dihydroxyindole-2-carboxylic acid from dopachrome. Like Palumbo etal, Leonard et al also do not teach or suggest that such metal ionscould be used to affect the color for formation of soluble melanin.

Many reports exist exploring the role of sulfhydryl compounds such ascysteine or glutathione in determining the red or yellow colors inmelanins (see review by Pawelek and Korner, "The Biosynthesis ofMammalian Melanin", American Scientist, 70, 136-145, 1982). Howeverthese reports do not teach or suggest that said sulfhydryl compoundscould be used to influence the colors of soluble melanin.

It would be of commercial value to have forms of melanin which aresoluble at physiological pH and temperature. Such melanins could beapplied evenly to mammalian skin and hair in appropriate vehicleswithout any of the caustic side-effects arising from the harsh reagentsneeded to solubilize precipitated melanins.

Such solubilized melanins could impart a naturally-appearing tan tomammalian skin and hair. Solubilized melanins would also be effective assun-screens, since melanins are the chemicals in the skin which absorbultraviolet radiation and thus provide protection from its harmfuleffects, such as premature skin aging and the occurrence of skincancers.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide solubilized formsof melanin at physiological pH and temperatures.

It is another object of the present invention to provide compositionsand methods for applying such melanins to mammalian skin and hair toprovide a naturally-appearing tan.

It is another object of the present invention to provide compositionsand methods for applying such melanins to mammalian skin and hair toprovide a sun-screen.

The above objects and other objects, aims, and advantages are satisfiedby the present invention. The present invention relates to a melaninthat is soluble in aqueous solution, e.g., water or an aqueous bufferedsolution, at a pH of at least 5 to 9, preferably 6.5 to 7.5, at atemperature of 0° to 100° C. The soluble melanin is furthercharacterized by being capable of being filtered through at least a 0.45micron size filter. The solubility of the melanin is in large part dueto the abundance of carboxyl-groups.

The present invention also concerns a method of producing solubilizedmelanin comprising combining in a reaction mixture dopachrome and one ormore enzymes derived from biological cells or tissues which have apigmentary system. 5,6-dihydroxyindole may be added to the dopachromeand enzyme(s) or dopachrome may be allowed to spontaneously form5,6-dihydroxyindole before adding the enzyme(s). Alternatively, thereaction mixture may comprise 5,6-dihydroxyindole-2-carboxylic acidalone or in a mixture with 5,6-hydroxyindole, in which case enzymes arenot necessary and the reaction occurs in the presence of oxygen.

The color of the soluble melanin can be varied between black, brown, redand yellow by altering the contents of the reaction mixtures, forexample by adding sulfhydryl containing compounds, or various metalssuch as, but not limited to, CU²⁺, Ni²⁺, and CO²⁺ or by altering the pHof the reaction mixtures.

The basis for the solubility of the melanin is in a large part due tothe high degree of carboxyl groups present in the molecule, saidcarboxyl groups being incorporated a part of the5,6-dihydroxyindole-2-carboxylic acid precursor. Compounds similar to5,6-dihydroxyindole-2-carboxylic acid could substitute in providing saidcarboxyl groups and could therefore also act as precursors to solublemelanin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a photograph showing the enzymatic formation of solublemelanin according to the invention. Non-enzymatically formed solublemelanin is similar in composition to that seen in the tubes labelled "DIComplex".

FIG, 2 depicts a graph showing the optical density (O.D.) of solublemelanin according to the invention at wavelengths greater than 300 nm.

The soluble melanin was synthesized non-enzymatically by mixing5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid(DHICA). The closed diamonds represent a fresh, non-incubated mixture ofDHI and DHICA where no soluble melanin was present. The open squaresrepresent the same mixture incubated 18 hours at room temperature in thepresence of oxygen during which time soluble melanin was synthesized.

The high absorbance peaking between 310-320 nm is characteristic of thepresence of DHICA in the melanin. The broad absorbance over the rangefrom 400-600 nm is due in the presence of visible color, characteristicof soluble melanins. Shown here are absorbance spectra in the"ultraviolet A" range and higher. Not shown are the strong absorbancespectra of the soluble melanin in the ultraviolet B and C ranges.

FIG. 3 depicts a graph showing the optical density (O.D.) of solublemelanin according to the invention at wavelengths greater than 300 nm.The soluble melanin was synthesized non-enzymatically as in FIG. 2 undereither aerobic (closed diamonds) or anaerobic conditions (open square).It can be seen that oxygen increases the amount of absorbance in the400-600 nm range, i.e., the amount of visible color.

FIG. 4 depicts a graph showing the optical density (O.D.) of solublemelanin according to the invention at wavelengths greater than 300 nm.The melanin was synthesized either non-enzymatically by mixing DHI andDHICA (closed diamonds) as described in FIGS. 2 and 3; or enzymaticallyby mixing dopachrome isomerase enzyme complex (DI) with dopachrome andDHI (open diamonds), or by mixing DI with only DHI (open squares), or bymixing DI with DHI plus DHICA (closed squares). The results demonstratethat enzymatic and non-enzymatic methods for synthesizing solublemelanin yield comparable products. They also demonstrate the necessityof DHICA or a compound similar to DHICA in the reaction mixtures.

FIG. 5 depicts a graph showing the optical density of soluble melaninsynthesized non-enzymatically at pH 7. The reaction mixtures werecomprised of a mixture of DHICA and DHI (closed diamonds) or DHICA alone(open squares). The results demonstrate that at ph 7 more visiblemelanin (400-600 nm) is synthesized with a mixture of DHI and DHICA thanwith DHICA alone.

FIG. 6 depicts a graph showing the optical density of soluble melaninsynthesized non-enzymatically at pH 8. The reaction mixtures areotherwise the same as those in FIG. 5. The results demonstrate that atpH 8, DHICA alone can serve as an efficient precursor to the formationof soluble melanin and in fact is somewhat superior to a mixture ofDHICA and DHI.

FIG. 7 depicts a graph showing a pH titration curve as increasingamounts of acetic acid are added to a solution of 2 mM soluble melanin.

FIG. 8 depicts a graph showing the precipitation of soluble melaninduring the pH titration shown in FIG. 7. For each point, acetic acid wasadded and the solution was allowed to sit at room temperature beforebeing filtered through a 0.45 micron filter. The optical density of thefiltrate was then determined at 500 nm. The soluble melanin begins toprecipitate below pH 4, i.e. as the pK of the carboxyl groups isreached. The results are consistent with the solubility of the melaninbeing determined by the number of non-protonated carboxyl groups presentin the molecule.

DETAILED DESCRIPTION OF THE INVENTION

The soluble melanin of the invention remains in aqueous solution, atneutral pH (e.g., pH of 5 to 9, preferably 6.5 to 7.5), for long periodsof time, e.g., indefinitely, at temperatures of 0° C. to 100° C., e.g.,room temperature. The soluble melanin according to the invention isfurther characterized by remaining soluble upon freezing/thawing. Theinventive soluble melanin is also characterized by being capable ofbeing filtered through at least a 0.45 micron size filter. The solublemelanin according to the invention can be precipitated below pH4.

Following synthesis, the soluble melanin cannot be dialyzed through asemi-permeable membrane which allows the passage of molecules less thana molecular weight of approximately 10,000 kilodaltons. Therefore thesoluble melanin according to the invention is of a molecular weightgreater than 10,000 kilodaltons, however, this is not an essentialcharacteristic for its usefulness. The soluble melanin can belyophilized to a dry powder form and then reconstituted to its solubleform with distilled water or suitable aqueous solvents, e.g., sodiumphosphate 0.1M or sodium chloride 0.1M.

The soluble melanin according to the invention can be preparednon-enzymatically (synthetically) or enzymatically.

The enzymatic preparation according to the invention comprises combiningin a reaction mixture a substrate, i.e., dopachrome, and one or moreenzymes derived from biological cells or tissues which contain apigmentary system and more particularly have the ability to producemelanin.

In the non-enzymatic preparation according to the invention, thereaction mixture comprises as a substrate5,6-dihydroxyindole-2-carboxylic acid (DHICA) alone or a mixture ofDHICA and 5,6-dihydroxyindole. Suitable analogs of DHICA, i.e. similarstructures containing carboxyl groups, maybe substituted in thereaction. The enzymatic or nonenzymatic reaction mixtures may stillfurther comprise as a substrate indole-5,6-quinone and/or melanochrome.Metal ions and sulfhydryl-containing compounds may be included.

The individual components of the substrate, be it one component, i.e.,dopachrome, as in the enzymatic preparation, or more than one componentas in the nonenzymatic preparation, preferably will be in an amount of0.01 to 5.0 millimolar. Stated otherwise, when more than one componentis used, the components, i.e., a mixture of 5,6-dihydroxyindole and5,6-dihydroxyindole-2-carboxylic acid, will preferably be in equalproportions or near to equal proportions.

The combining of substrate and enzymes or substrates in the reactionmixture is preferably conducted at a temperature of 15° C. to 37° C.

It is preferred in both the enzymatic and nonenzymatic preparation thatoxygen, e.g., air or pure oxygen, be present. This is especially truefor the nonenzymatic preparations.

Structural formulas and the relationship among some of the abovedescribed compounds are depicted as follows: ##STR1##

From the above it is seen that dopa quinone, leuco dopachrome,dopachrome, DHICA, 5,6-dihydroxyindole, indole-5,6-quinone, andmelanochrome are all derivatives of dopa. Dopa itself is a derivative oftyrosine, so the above compounds are also derivatives of tyrosine (seeScheme II). Scheme I shows that dopachrome can give rise to5,6-dihydroxyindole in a spontaneous non-enzymatic reaction, or it cangive rise to DHICA in an enzymatically catalyzed reaction. The enzymewhich catalyzes dopachrome to DHICA is named dopachrome isomerase andmay indeed be the enzyme responsible for soluble melanin formation. Theenzyme may also be a part of a complex comprising tyrosinase, dopachromeisomerase, glycoprotein 75, MSH receptor and other unknown proteins.

The above described enzymes are described in the following papers:

tyrosinase: Ann Korner and John Pawelek, "Mammalian Tyrosinase CatalyzesThree Reactions in the Biosynthesis of Melanin", Science, 217:1163-1165,1982;

dopachrome isomerase: John Pawelek, "Dopachrome Conversion FactorFunctions as an Isomerase", Biochemical and Biophysical ResearchCommunications, 166:1328-1333, 1990;

glycoprotein 75: Timothy M. Thomson, M. Jules Mattes, Linda Roux, LloydOld and Kenneth O. Lloyd, "Pigmentation-associated Glycoprotein of HumanMelanomas and Melanocytes: Definition with a Mouse Monoclonal Antibody",J. Invest. Derm., 85:169-174, 1985;

MSH receptor: Seth J. Orlow, Sara Hotchkiss, and John M. Pawelek,"Internal Binding Sites for MSH: Analyses in Wild-Type and VariantCloudman Melanoma Cells", J. Cellular Physiology, 142:129-136, 1990.

The enzyme may also include dopachrome isomerase and one or more ofglycoprotein 75, MSH receptor and tyrosinase.

The enzyme may take the form of one or more individual enzymes or acomplex of enzymes including tyrosinase, dopachrome isomerase,glycoprotein 75, MSH receptor and one or more additional enzymes whichare distinct from the aforesaid four described enzymes, but which arecapable of catalyzing the synthesis of soluble melanin.

The soluble melanin according to the present invention can be admixedwith a physiologically acceptable carrier to form a composition.

Physiologically acceptable carriers useful in the practice of theinvention are known in the art and non-limiting examples of suchcarriers include, for controlled release--microcapsules comprisingcarboxymethylene copolymers; for transdermal release--acrylamides andfor topical application--cosmetic bases.

In addition, if desired, the composition according to this embodimentcomprises at least one additive selected from the group consisting ofsolvents, fragrances, sunscreening agents, preservatives and chelatingagents.

Cosmetic bases useful in the practice of the invention are well knownand include lotions, creams, ointments and dusting powders. Examplesthereof may be found in, e.g., U.S. Pat. Nos. 4,228,151; 4,282,206 and2,949,403.

Solvents for use in accordance with the invention include, for example,ethanol, isopropyl alcohol, benzyl alcohol, oils, for example, groundnut oil, distilled and/or deionized-water, physiological saline solutionand the like. The specific solvent chosen will depend on the method ofapplication.

It may also be desirable to add a preservative to the inventivecompositions if they are to be used for topical applications. Thepreferred mode of administration of the inventive compositions istopical administration. Still further, the soluble melanin of thepresent invention may be combined with substances that stimulate thepigmentary system under conditions of low levels of UV light.

Preservatives are well known and may be exemplified by methylparaben,"DOWACIL 2000" and propylparaben.

If desired, in order to reduce the acidity or basicity of the inventivecompositions, bases, acids or buffers may be added thereto in accordancewith the knowledge of the art.

The concentration of soluble melanin in an aerosol, cream, lotion orother composition is preferably 0.01 mg/ml to 1.0 mg/ml.

Solutions have different colors depending on the concentration of"chromophore" dissolved in them. For example, a deep red solution willappear orange or pink when diluted with more solvent, but no additionalchromophore. In the case of the soluble melanin, when it is dissolved ata fairly high concentration in water, e.g., 0.5 mg/ml, it appearsbrown-black in color. When more water is added so that the concentrationof the soluble melanin is reduced to, e.g., 0.1 mg/ml, the solutionappears golden in color. It is not believed that diluting the materialchanges any shift in the absorbance spectrum, rather it is believed tobe a visual perception.

For the nonenzymatic preparation, the 5,6-dihydroxyindole-2-carboxylicacid and 5,6-dihdyroxyindole may be maintained separately, for example,in microspheres, or in separate tubes or containers, until being mixedtogether on the skin of a mammal, e.g., human.

The invention will now be described with reference to the followingnon-limiting examples.

EXAMPLE 1 Source of Enzymes

The enzyme preparation for the synthesis of soluble melanin can beobtained from any biological cells or tissues which have the ability toproduce melanin, for example:

(1) extracts of vertebrate skins from horses, cattle, sheep, pigs, orany other such mammalian source; extracts of skins of fish, amphibia,reptiles, birds, or any other such vertebrate source;

(2) extracts of any botanical source which has the ability to producemelanin such as mushrooms, potatoes, bananas, or any other suchbotanical source;

(3) extracts of invertebrate organisms such as worms, arthropods, or anysuch invertebrate source which has the ability to produce melanin;

(4) extracts of any single-cell organisms which have the ability toproduce melanin, but which do not necessarily fall into a category ofzoological or botanical, such as bacteria and protozoans;

(5) extracts of any organisms in which genes for enzymes producingsoluble melanin have been genetically cloned; and

(6) culture media of any organisms or cells which secrete enzymes forproducing soluble melanin--such organisms or cells may or may notexpress cloned genes for the enzymes.

Furthermore, the source of biological material for the isolation ofenzymes to produce soluble melanin does not necessarily have to beactively synthesizing melanin in vivo. A pigmentary system is defined asall or part of a group of enzymes which can recognize as theirsubstrates precursors and/or intermediates in the melanin biosyntheticpathway. Some biological sources contain a pigmentary system, but do notsynthesize melanin, however, extracts derived from them can producesoluble melanin. These biological systems, in their living state, arereferred to as "amelanotic", or "non-pigmented". Many albino organismsfall into this category, i.e., they possess an incomplete or inhibitedpigmentary system and do not make melanin in vivo, however, extractsfrom some albino organisms contain enzymes that can produce solublemelanin. Such organisms are also potential sources of enzymes for theproduction of soluble melanins.

EXAMPLE 2 Preparation of Enzymes

The enzymes for production of soluble melanin can be isolated from anextract of an appropriate biological source, or, from the culture mediashould the enzymes be secreted into the media by the source (see Example1). Extracts are prepared by lysing the cells of the biological sourcethrough procedures such as homogenization (e.g., in a common kitchen"blender", in appropriate glass, metal, or plastic tissue homogenizers);such as freeze-thawing in a hypotonic solution such as water; or by anymeans which disrupt the cellular wall or plasma membrane of thebiological source. In cases where the enzymes for producing melanin arethemselves insoluble (e.g., in a particulate form within the cells), itmay be necessary to lyse the cells in the presence of a non-ionicdetergent such as "TRITON X-100", "CHAPS", or "TWEEN 80", or an organicsolvent such as acetone or ethanol, or any other solvents whichsolubilize the enzymes, without destroying their ability to producesoluble melanin.

The following procedures may be useful, but are not mandatory for thepreparation:

Extracts containing the enzymes in a soluble form can be clarified byfiltration through such material as gauze or filter paper; or bycentrifugation; or by "settling" through the use of naturalgravitational force.

Extracts can be further clarified by mixing them with calcium phosphate(also known as "hydroxylapatite") which is itself insoluble, but whichattaches to many molecular structures in cells, but does not attach tothe enzymes for preparation of soluble melanins. The calcium phosphateand its attached molecules can be removed from the extract byfiltration, centrifugation, or gravitational settling as describedabove. Although calcium phosphate is useful in this regard, theprocedure is not restricted to the use of this agent only. Any insolublecompound which attaches to molecular structures other than the enzymesin question and does not destroy the activity of the enzymes can beemployed.

Extracts can be further clarified by mixing them with an anion exchangeagent such as diethylaminoethyl cellulose. At conditions around neutralpH (e.g., pH 6.5-7.5) and low buffer concentration (e.g., 5-10millimolar sodium phosphate), the enzymes for production of solublemelanin will attach to such an anion exchange agent, while many othermolecular structures will not. The enzymes in question can then beeluted from the anion exchange agent by increasing the saltconcentration (e.g., by adding 0.4 molar sodium chloride, or by using agradient of sodium chloride from 0 molar to 0.4 molar). Althoughdiethylaminoethyl cellulose, sodium phosphate, and sodium chloride areuseful in this regard, the procedure is not restricted to the use ofthese agents only. Any anion exchange resin, buffer, or salt which doesnot destroy the activity of the enzymes in question can be employed. Theprinciple is the same.

Extracts can be further clarified by mixing them with an agent whichattaches to glycoproteins, such as wheat germ lectin-sepharose. Inmammalian cells, the enzymes in question are glycoproteins and thereforeattach to such lectins, while many other molecular structures do not.The enzymes in question can then be eluted from the lectin by mixingwith an appropriate sugar such a N-acetylglucoseamine, which causes adisplacement of the enzymes from the lectin. Although wheat germlectin-sepharose, and N-acetylglucoseamine are useful in this regard,the procedure is not restricted to the use of these agents only. Anyappropriate lectin or sugar which does not destroy the activity of theenzymes in question can be employed. The principle is the same.

Extracts can be further clarified by applying them to columns containingmolecular sieves such as Sephadex columns, or high pressure liquidchromatography columns containing molecular sieves. The enzymes inquestion can be eluted from such columns with non-destroying buffersaccording to their molecular weights and can be thereby separated fromother molecular structures with differing molecular weights. AlthoughSephadex columns and various HPLC resins are useful molecular sieves,the procedure is not restricted to the use of these agents only. Anyappropriate method for separation of molecules according to theirmolecular weights which does not destroy the activity of the enzymes inquestion can be employed. When each of the above procedures is carriedout in the sequence listed, a preparation is obtained which contains thefollowing: tyrosinase, dopachrome isomerase, a protein designated"glycoprotein 75" or "gp75", which exhibits catalase activity, and aprotein designated "MSH receptor". That is, these four known proteinsco-purify through the above procedures. Analyses by polyacrylamide gelelectrophoresis indicate that there may be ten or more proteins intotal. It is not presently known which protein or combination thereofcatalyzes the synthesis of soluble melanin, although the synthesis canoccur in the presence of phenylthiourea, a potent inhibitor oftyrosinase, suggesting that tyrosinase may not be necessary.

EXAMPLE 3 Enzymatic Synthesis of Soluble Melanin

Soluble melanin is prepared enzymatically by mixing the enzymes,isolated and purified as described above, with a solution containingdopachrome and 5,6-dihydroxyindole. The enzymes and the substrates areallowed to incubate in a non-destroying buffer (e.g., 0.1 molar sodiumphosphate, pH 6.5-7.5) at ambient temperature or any suitablenon-destroying temperature which allows for the reaction to occur (e.g.,15°-37° C.), until soluble melanins begin to appear (e.g., 3-6 hours).The reaction can be monitored visually or with the use of aspectrophotometer set in the visual spectrum (e.g., 400 millimicrons).When the reaction has reached completion, the salts from the buffer canbe removed by dialysis and the soluble melanin can be stored at roomtemperature, frozen, or as a crystal or powder obtained through suchprocedures as natural evaporation or lyophilization. It is useful, butnot mandatory, to enzymatically synthesize soluble melanin in thepresence of a tyrosinase inhibitor such as phenylthiourea, becausetyrosinase, which is occasionally present in the enzyme preparation, cancause the formation of insoluble melanin. Although phenylthiourea is auseful tyrosinase inhibitor because it does not inhibit or destroy theenzymes in question, the procedure is not restricted to phenylthioureaand any such tyrosinase inhibitor can be employed.

Soluble melanin prepared by the above procedure is golden-brown in colorand absorbs widely throughout the ultraviolet and visible spectra (e.g.,220 to 700 millimicrons). The color can vary from brown-black in veryconcentrated solutions, to golden in more dilute solutions. The additionof sulfhydryl-containing compounds such as cysteine or glutathione canimpart a reddish color to the soluble melanins.

FIG. 1 depicts the enzymatic formation of soluble melanin. The contentsof the three tubes on the right were filtered through a 0.45 micronfilter, otherwise they were identical in composition to the contents ofthe three tubes on the left. Considering the three tubes on the left:

1) The far left-hand tube contained a mixture of dopachrome, dopa and5,6-dihydroxyindole. It also contained the buffer used to dissolve theenzymes (sodium phosphate 5 mM, pH 6.8, containing 20% glycerolvol/vol), but the enzymes themselves were not added.

2) The second tube from the left contained the same mixture as in theleft-hand tube, but in addition it contained the enzymes dissolved intheir buffer. The enzymes are referred to as "DI Complex".

3) The third tube from the left was identical to the middle tube, butalso contained trypsin, a potent proteolytic enzyme, at a concentrationof 0.5 mg/ml.

The three-tubes on the left were incubated at room temperature for 6hours before half their contents were removed and filtered as describedabove into the three right-hand tubes.

It can be seen that with buffer only, all the black melanin-which formedwas trapped on the filter, i.e., insoluble. When the DI Complex wasadded, the golden-brown melanin was completely filterable, i.e.,soluble. When trypsin was present, no soluble melanin was formed,indicating that the soluble melanin production was catalyzed by aprotein (enzyme). Melanins prepared non-enzymatically are similar inappearance to those seen in the tubes labelled "DI Complex".

The soluble melanins provided in Example 3 have the followingcharacteristics:

(1) are greater than molecular weight 10,000,

(2) are stable to boiling,

(3) are stable to freezing,

(4) can be filtered through a filter at least as small as 0.45 microns,

(5) are soluble in water at a pH range of at least 6.5 to 7.5 attemperatures from 0° to 100° C.,

(6) can be precipitated below pH 4,

(7) can be lyophilized to a crystal/powder form which can be redissolvedin water,

(8) vary from brown-black to golden in color depending on concentration,

(9) absorb throughout the ultraviolet and visible spectra and

(10) can be prepared in red and yellow forms with the addition ofsulfhydryl-containing compounds and various metal ions.

EXAMPLE 4 Non-Enzymatic Synthesis of Soluble Melanin

Soluble melanin can be prepared in a nonenzymatic reaction by mixing5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-hydroxyindole(DHI) in the presence of oxygen in a non-destroying buffer (e.g., 0.1molar sodium phosphate, pH 6.5 to 7.5) or by incubating DHICA alone atambient temperature of any suitable non-destroying temperature whichallows the reaction to occur (e.g., 15 to 37° C.), until soluble melaninbegins to appear (e.g., 3 to 6 hours). The soluble melanins thus formedis indistinguishable from that which is formed enzymatically asdescribed in Example 3.

The difference between enzymatic and non-enzymatic synthesis of solublemelanin is that in the enzymatic synthesis,5,6-dihydroxyindole-2-carboxyl acid is produced from dopachrome by theenzyme dopachrome isomerase, and 5,6-dihydroxyindole is producedspontaneously from dopachrome, while in the non-enzymatic synthesis,5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindoleare mixed directly to form soluble melanin or DHICA is incubated alone.In both the enzymatic and non-enzymatic procedures, the reaction isgreatly enhanced by the presence of oxygen.

EXAMPLE 5 Spectrophotometric Quantitation of Soluble Melanin

Melanin was synthesized using a mixture of L-dopa, dopachrome,dihydroxyindole, and dihydroxyindole-2-carboxylic acid at concentrationsof approximately 0.4 mg/ml dissolved in sodium phosphate, 0.1M, pH 6.8.The "DI Complex" enzymes purified approximately 2,000 fold from 0.5grams mouse melanoma tissue (see Example 3) were dissolved in buffer(sodium phosphate, 5 mM, pH 6.8) and incubated in a 4 ml reaction mixwith the above substrates at room temperature for 5 hours. The controlreaction had buffer only, with no enzymes added. In both reactions,melanin formation occurred, but when the enzymes were present, themelanin could be filtered through a 0.45 micron filter, whereas in thepresence of buffer only, the melanin was insoluble and could not befiltered. Solutions were diluted 50 fold before measuring the opticaldensity. A photographic representation of this experiment is seen inFIG. 1.

FIGS. 2 to 4 show the optical spectrum of soluble melanin according tothe present invention. In FIGS. 2 to 4, the melanin exhibited a peakoptical density (O.D.) at a wavelength of 310-320 nm. The spectra andO.D. were the same after the melanin had incubated under sterileconditions for 2 months at room temperature (20° C.), and whether or notthe melanin was dialyzed or filtered through a 0.45 micron filter.

Note in FIG. 4 that relatively little soluble melanin is made when DHIplus DI are mixed together in the absence of any other added substrates.Note also, when DHI plus DHICA are mixed together they make as muchsoluble melanin as when they are mixed additionally with DI, i.e., DI isnot necessary in this case. Finally, note that a mixture of dopachromeand DHI in the absence of DI results in an insoluble precipitate (notshown here), but in the presence of DI results in the synthesis ofsoluble melanin. This is because DI converts dopachrome to DHICA whichthen in turn combines with DHI in the presence of oxygen to form solublemelanin. In the absence of DI, dopachrome spontaneously converts to DHIand a precipitate (insoluble melanin) forms (see Scheme I hereinabove).

FIGS. 5 through 8 are described hereinabove.

                  TABLE 1                                                         ______________________________________                                        Spectrophotometric Quanititation of Soluble Melanin                                       Optical Density at Wavelength 320 nm                              Synthetic Route                                                                             Before Filtration                                                                          After Filtration                                   ______________________________________                                        Buffer only   1.22          .024                                              (no enzymes added)                                                            Enzymes added 0.982        0.966                                              ______________________________________                                    

EXAMPLE 6 Transdermal Release Composition

An admixture is prepared comprising the following:

    ______________________________________                                                         Parts by weight                                              ______________________________________                                        Acrylamide copolymer                                                                             20                                                         (e.g., "polytrap FLME 203")                                                   soluble melanin, as prepared                                                                     5                                                          according to Examples 3 or 4                                                  Alcohol            74.9                                                       and a fragrance    0.1                                                        ______________________________________                                    

The above mixture is applied to the skin, once a day, preferably in themorning, for two to four weeks.

EXAMPLE 7 Tanning Oil

A. An admixture is prepared by adding in the order indicated:

    ______________________________________                                                           Parts by weight                                            ______________________________________                                        decylolcate          25.0                                                     isopropyl myristate  15.0                                                     and propylene glycol dicaprylate/                                                                  5.0                                                      dicaprate                                                                     mineral oil          54.85                                                    ______________________________________                                    

B. An admixture is prepared by adding 0.01 pbw soluble melanin, e.g., asprepared as described in Example 3 or 4, to 0.01 pbw of "SOLERTAN PB-10"(a poly(propylene glycol) lanolin ether).

C. The admixture of part B is added to the admixture of part A and theresultant admixture is mixed until homogeneous.

D. The composition of part C is applied to the skin once or twice dailyfor two to four weeks.

EXAMPLE 8 Suntanning Lotion

An admixture was prepared containing the following:

    ______________________________________                                                         Parts by weight                                              ______________________________________                                        ICI G-1800         5.0                                                        (e.g., poly[oxyethylene]21                                                    stearyl ether)                                                                isopropyl myristate                                                                              10.0                                                       preservative       0.1                                                        stearyl alcohol    2.0                                                        2-hydroxy-3,3,5-   8.0                                                        trimethylhexyl                                                                ester of benzoic acid                                                                             0.05                                                      butylated hydroxyanisole                                                      ______________________________________                                    

The above mixture is heated to 70° C. and 60 parts by weight of water,preheated to 70° C. is added thereto. the resultant mixture is stirredand allowed to cool to room temperature.

To the above mixture is then added a 1% citric acid solution, QS, toachieve a pH of 5.0 after which 0.01 parts by weight of a solublemelanin, for example, as prepared according to Example 3 or Example 4,is added, as well as sufficient deionized water to yield 100 parts byweight of lotion.

The above lotion is applied to the skin one-half (1/2) hour prior toexposure to the sun. After swimming, sweating or toweling, as well asafter each hour of exposure, the lotion is reapplied.

It will be appreciated that the instant specification is set forth byway of illustration and not limitation, and that various modificationand changes may be made without departing from the spirit and scope ofthe present invention.

What is claimed is:
 1. A composition for a sunscreen for mammalian skinor hair or for providing a naturally-appearing tan to mammalian skincomprising an aqueous soluble melanin at a concentration of about 0.01to 1.0 mg/ml, the soluble melanin having been produced by polymerizing adihydroxyindolecarboxylic acid in an aqueous medium with aeration.
 2. Amethod of providing a naturally-appearing tan to mammalian skincomprising topically applying to a mammal's skin an effective tanproducing amount of an aqueous soluble melanin at a concentration ofabout 0.01 to 1.0 mg/ml, the soluble melanin having been produced bypolymerizing a dihydroxyindolecarboxylic acid in an aqueous medium withaeration.
 3. A method of providing a sun screen to mammalian skin andhair comprising topically applying to a mammal's skin or hair aneffective sun screening amount of an aqueous soluble melanin at aconcentration of about 0.01 to 1.0 mg/ml, either alone, or in admixturewith a physiologically acceptable carrier in the form of an oil, creamor ointment, the soluble melanin having been produced by polymerizing adihydroxyindolecarboxylic acid in an aqueous medium with aeration.